Complex and rapidly adaptable regulatory networks allow bacteria such as E. coli to change metabolism to optimize growth and survival, both aerobically and anaerobically, in mammalian hosts and outside of the host and in response to a variety of stresses. In the last twenty years, the important roles of small non-coding RNAs in regulation in all organisms have been recognized. Our laboratory, in collaboration with others, undertook two global searches for non-coding RNAs in E. coli, contributing significantly to the 100-200 regulatory RNAs that are now identified. A large number of these small RNAs (sRNAs) bind tightly to the RNA chaperone Hfq. We and others have shown that sRNAs that binds tightly to Hfq act by pairing with multiple target mRNAs, regulating stability and translation of the mRNA, either positively or negatively, although some of these sRNAs also have additional roles. Our lab has studied many of these sRNAs in detail. Each sRNA is regulated by different stress conditions, suggesting that the sRNA plays an important role in adapting to stress. We have also examined the mechanism by which Hfq operates to allow sRNAs to act. The lab continues to investigate the in vivo roles of small RNAs, identifying the regulatory networks they participate in and their roles in those networks. Using approaches for screening targets of interest and the sRNAs regulating them, previously developed in the laboratory, we continue to investigate regulatory pathways for sRNAs. mutS, encoding a component of the mismatch repair system, was found to be regulated by a small RNA, ArcZ, and, somewhat surprisingly, directly by Hfq in the absence of sRNAs, dependent upon sites in the mutS 5'UTR. Mutation of these sites leads to increased levels of MutS protein in stationary phase cells and decreased mutagenesis, demonstrating the role of post-transcriptional regulation in allowing mutagenesis as cells run out of nutrients. In another project, a small RNA processed from the 3' UTR of an operon encoding TCA proteins has been found to regulate levels of the signaling molecule acetyl phosphate and change flux through the acetate switch. Lessons learned from this project suggest the importance of many other previously unappreciated sRNAs made from 3' UTRs. The action of these small RNAs depends on the RNA chaperone Hfq, a protein with homology to the Lsm and Sm families of eukaryotic proteins involved in RNA splicing and other functions. Hfq binds both to sRNAs and to mRNAs, and stimulates pairing, but exactly how it does this has been clear. In a series of studies, in collaboration with G. Storz (NICHD) and with S. Woodson (JHU), we have carried out an in vivo dissection of Hfq that has changed our understanding of how this protein acts with sRNAs. We have found that the Hfq-dependent sRNAs fall into two classes, defined by their behavior in different Hfq mutants. All of these sRNAs depend on the known sRNA binding site on the proximal face of Hfq for in vivo stability. Class I sRNAs are rapidly degraded when used, most likely dependent upon pairing; their targets bind to the distal face. Class II sRNAs are generally more stable than Class I sRNAs, and their targets bind to rim sites in Hfq. These results help to explain previously observed competition between sRNAs and differential effects of different hfq alleles on different sRNA:mRNA pairs. The C-terminus of E. coli Hfq (CTD) is unstructured, and its role has been unclear. In collaboration with S. Woodson, we have defined in vivo and in vitro roles for the CTD in stabilization and release of Class II sRNAs. In recent work in our lab, we have, in collaboration with G. Storz, examined the global effect of deleting the CTD of Hfq, and find only subtle effects on RNA accumulation. However, in combination with mutations on the RNA binding faces of Hfq, loss of the CTD can have synergistic effects that should give new insight into its role. Using a newly developed bi-functional fluorescent reporter we have identified novel regulators of sRNA stability and function, including a new RNA sponge and two previously uncharacterized proteins, one of which has global effects on sRNA-based regulation and another of which has effects on the stability of specific sRNAs. Each of these opens up previously unknown levels of regulation of sRNA function. Overall, we have developed highly efficient in vivo tools for studying sRNAs and the networks they reside in. Our focus is increasingly on the role of the sRNAs in complex bacterial behavior, investigations into the mechanism of sRNA function, and dissecting of novel mechanisms for regulating translation initiation. We identify genetic changes (mutations or overexpression) that perturb expression of the RpoS general stress factor, and use those to investigate regulatory pathways. For instance, H. Tabor's lab (NIDDK) had observed that cells devoid of polyamines have very low levels of RpoS; in a collaboration with them, we have confirmed this work and are identifying the pathways that decrease RpoS accumulation. We have also returned to our interest in the regulatory cascade affecting capsule synthesis, in a collaboration with S. Buchanan and NCATs. The proteins in this cascade also regulate aspects of the bacterial response to membrane stress, are needed for in vivo establishment of commensal growth, and are important virulence factors in Klebsiella. Studies on the Interactions of the components of the regulatory cascade have changed our understanding of signal transduction through this system, demonstrating that a critical negative regulator of signaling acts by interaction with a phosphorelay protein, leading to a major revision in our understanding of signaling in this system and providing new insight into the general principles affecting related and widespread signaling systems. We have developed an efficient assay for screening for small molecules that activate or inactivate the cascade and have found evidence for effects of a variety of antibiotics in inducing the system. The long-term goal of this is to investigate the development of novel antibiotics that act by perturbing this important regulon.